Basement membrane remodeling in skeletal muscles of patients with limb ischemia involves regulation of matrix metalloproteinases and tissue inhibitor of matrix metalloproteinases

BACKGROUND/AIM: Because the pericapillary basement membrane in skeletal muscles of patients with chronic critical limb ischemia (CLI) is thickened, we determined the expression patterns of genes involved in collagen metabolism, using samples from 9 CLI patients, 4 patients with acute limb ischemia and 4 healthy controls. METHODS: Gene array analysis, quantitative RT-PCR and semiquantitative grading of immunohistochemical reactivity were performed to determine mRNA/cDNA and protein concentrations. RESULTS: In CLI patients compared to controls, cDNA levels of matrix metalloproteinase (MMP)-9 and MMP-19 were higher, collagen type IV chains A1 and A2, tissue inhibitor of matrix metalloproteinase (TIMP)-1 and TIMP-2 were similar and MMP-2 were lower. On the protein level, MMP-2, MMP-9, MMP-19 and TIMP-1 were more abundantly expressed. In skeletal muscles from patients with acute limb ischemia, cDNA and protein levels of MMP-9, MMP-19, collagen type IV chains, TIMP-1 and TIMP-2 were high. MMP-2 was elevated at the protein but decreased on the cDNA level. CONCLUSION: Expression of basement membrane components in skeletal muscles of CLI and acute limb ischemia patients is altered, possibly contributing to the pathogenesis of peripheral arterial disease.

Attenuation of myocardial reperfusion injury in pigs by Mirococept, a membrane-targeted complement inhibitor derived from human CR1

OBJECTIVES: Membrane-targeted application of complement inhibitors may ameliorate ischemia/reperfusion (I/R) injury by directly targeting damaged cells. We investigated whether Mirococept, a membrane-targeted, myristoylated peptidyl construct derived from complement receptor 1 (CR1) could attenuate I/R injury following acute myocardial infarction in pigs. METHODS: In a closed-chest pig model of acute myocardial infarction, Mirococept, the non-tailed derivative APT154, or vehicle was administered intracoronarily into the area at risk 5 min pre-reperfusion. Infarct size, cardiac function and inflammatory status were evaluated. RESULTS: Mirococept targeted damaged vasculature and myocardium, significantly decreasing infarct size compared to vehicle, whereas APT154 had no effect. Cardioprotection correlated with reduced serum troponin I and was paralleled by attenuated local myocardial complement deposition and tissue factor expression. Myocardial apoptosis (TUNEL-positivity) was also reduced with the use of Mirococept. Local modulation of the pro-inflammatory and pro-coagulant phenotype translated to improved left ventricular end-diastolic pressure, ejection fraction and regional wall motion post-reperfusion. CONCLUSIONS: Local modification of a pro-inflammatory and pro-coagulant environment after regional I/R injury by site-specific application of a membrane-targeted complement regulatory protein may offer novel possibilities and insights into potential treatment strategies of reperfusion-induced injury.

Outer membrane protein UspA1 and lipooligosaccharide are involved in invasion of human epithelial cells by Moraxella catarrhalis

Invasion of non-professional phagocytes is a strategy employed by several mucosal pathogens, but has not been investigated in detail for Moraxella catarrhalis, a major cause of human respiratory tract infections. We investigated the role of outer membrane protein (OMP) UspA1 and lipooligosaccharide (LOS) in M. catarrhalis invasion into epithelial cells. An isogenic mutant of strain O35E, which lacked expression of the UspA1 adhesin, demonstrated not only severely impaired adherence (86%) to but also reduced invasion (77%) into Chang conjunctival cells in comparison with the wild-type strain. The isogenic, LOS-deficient mutant strain O35E.lpxA was attenuated in adherence (93%) and its capacity to invade was severely reduced (95%), but not abolished. Inhibition assays using sucrose and cytochalasin D, respectively, demonstrated that clathrin and actin polymerization contribute to internalization of M. catarrhalis by Chang cells. Furthermore, inhibition of UspA1-mediated binding to cell-associated fibronectin and alpha5beta1 integrin decreased invasion of M. catarrhalis strain O35E (72% and 41%, respectively). These data indicate that OMP UspA1 and LOS profoundly affect the capacity of M. catarrhalis to invade epithelial cells.

Human immunodeficiency virus type 1 and influenza virus exit via different membrane microdomains

Directed release of human immunodeficiency virus type 1 (HIV-1) into the cleft of the virological synapse that can form between infected and uninfected T cells, for example, in lymph nodes, is thought to contribute to the systemic spread of this virus. In contrast, influenza virus, which causes local infections, is shed into the airways of the respiratory tract from free surfaces of epithelial cells. We now demonstrate that such differential release of HIV-1 and influenza virus is paralleled, at the subcellular level, by viral assembly at different microsegments of the plasma membrane of HeLa cells. HIV-1, but not influenza virus, buds through microdomains containing the tetraspanins CD9 and CD63. Consequently, the anti-CD9 antibody K41, which redistributes its antigen and also other tetraspanins to cell-cell adhesion sites, interferes with HIV-1 but not with influenza virus release. Altogether, these data strongly suggest that the bimodal egress of these two pathogenic viruses, like their entry into target cells, is guided by specific sets of host cell proteins.

Salivary antibodies directed against outer membrane proteins of Moraxella catarrhalis in healthy adults

Moraxella catarrhalis is a major mucosal pathogen of the human respiratory tract, but the mucosal immune response directed against surface components of this organism has not been characterized in detail. The aim of this study was to investigate the salivary immunoglobulin A (IgA) response toward outer membrane proteins (OMP) of M. catarrhalis in healthy adults, the group of individuals least likely to be colonized and thus most likely to display mucosal immunity. Unstimulated saliva samples collected from 14 healthy adult volunteers were subjected to IgA immunoblot analysis with OMP preparations of M. catarrhalis strain O35E. Immunoblot analysis revealed a consistent pattern of IgA reactivity, with the appearance of five major bands located at >250, 200, 120, 80, and 60 kDa. Eleven (79%) of 14 saliva samples elicited reactivity to all five bands. Immunoblot analysis with a set of isogenic knockout mutants lacking the expression of individual OMP was used to determine the identities of OMP giving rise to IgA bands. Human saliva was shown consistently to exhibit IgA-binding activity for oligomeric UspA2 (>250 kDa), hemagglutinin (200 kDa), monomeric UspA1 (120 kDa), transferrin-binding protein B (TbpB), monomeric UspA2, CopB, and presumably OMP CD. TbpB, oligomeric UspA2, and CopB formed a cluster of bands at about 80 kDa. These data indicate that the human salivary IgA response is directed consistently against a small number of major OMP, some of which are presently considered vaccine candidates. The functional properties of these mucosal antibodies remain to be elucidated.

Comparing oxygen transfer performance between three membrane oxygenators: effect of temperature changes during cardiopulmonary bypass

Recently, a new oxygenator (Dideco 903 [D903], Dideco, Mirandola, Italy) has been introduced to the perfusion community, and we set about testing its oxygen transfer performance and then comparing it to two other models. This evaluation was based on the comparison between oxygen transfer slope, gas phase arterial oxygen gradients, degree of blood shunting, maximum oxygen transfer, and diffusing capacity calculated for each membrane. Sixty patients were randomized into three groups of oxygenators (Dideco 703 [D703], Dideco; D903; and Quadrox, Jostra Medizintechnik AG, Hirrlingen, Germany) including 40/20 M/F of 68.6 +/- 11.3 years old, with a body weight of 71.5 +/- 12.1 kg, a body surface area (BSA) of 1.84 +/- 0.3 m(2), and a theoretical blood flow rate (index 2.4 times BSA) of 4.4 +/- 0.7 L/min. The maximum oxygen transfer (VO(2)) values were 313 mL O(2)/min (D703), 579 mL O(2)/min (D903), and 400 mL O(2)/min (Quadrox), with the D903 being the most superior (P < 0.05). Oxygen (O(2)) gradients were 320 mm Hg (D703), 235 mm Hg (D903), and 247 mm Hg (Quadrox), meaning D903 and Quadrox are more efficient versus the D703 (P < 0.05). Shunt fraction (Qs/Qt) and diffusing capacity (DmO(2)) were comparable (P = ns). Diffusing capacity values indexed to BSA (DmO(2)/m(2)) were 0.15 mL O(2)/min/mm Hg/m(2) (D703), 0.2 mL O(2)/min/mm Hg/m(2) (D903), and 0.18 mL O(2)/min/mm Hg/m(2) (Quadrox) with D903 outperforming D703 (P < 0.0005). During hypothermia (32.0 +/- 0.3 degrees C), there was a lower absolute and relative VO(2 )for all three oxygenators (P = ns). The O(2) gradients, DmO(2) and DmO(2)/m(2), were significantly lower for all oxygenators (P < 0.01). Also, Qs/Qt significantly rose for all oxygenators (P < 0.01). The oxygen transfer curve is characteristic to each oxygenator type and represents a tool to quantify oxygenator performance. Using this parameter, we demonstrated significant differences among commercially available oxygenators. However, all three oxygenators are considered to meet the oxygen needs of the patients.

Follicle-forming cat thyroid cell lines synthesizing extracellular matrix and basal membrane components: a new tool for the study of thyroidal morphogenesis

Interactions between follicular epithelial cells and extracellular matrix (ECM) are supposed to play an important role in the development and maintenance of thyroid tissue architecture. In the present study we have therefore investigated the synthesis of ECM components by a feline thyroid cell line which is able to form follicle-like structures in vitro, and also in v-ras-transfected and control-transfected sublines. Transfections were performed by lipofection with pZSR (viral Harvey ras gene; neo) and pSV2-neo (control, neo only) plasmids. We have adapted a semisolid culture system composed exclusively of polymerized alginate and therefore devoid of ECM components. Feline cells embedded in alginate gels as single cells and cultured for up to 90 days formed cell clusters within 10 days. Follicle-like structures were formed in the original cell lines and also in the v-ras- and control-transfected cells. Differences in proliferation rates were observed, the v-ras-transfected cells growing up to two to three times faster than the non-transfected cells. Immunostaining was done using rabbit first antibodies directed against mouse collagen IV, human fibronectin, laminin (tumor Engelbreth-Holm-Swarm laminin), perlecan and other ECM components. For comparison, immunostaining was also performed on cryosections of nodular goiters of six hyperthyroid cats. The cell lines and their transfected clones stained strongly positive for collagen IV and fibronectin, and positively but less strongly for laminin and perlecan. The cat goiter tissue stained positively for collagen IV, laminin, perlecan, and fibronectin, and positive staining for S-laminin (containing the beta2-chain) was seen in blood vessel walls in this tissue. In conclusion, cat cell lines grow three-dimensionally in alginate beads over several weeks, they form follicle-like structures and express the same ECM components as the native cat goiter tissue. Transfection with v-ras does increase proliferation rate, but does not fundamentally alter formation of follicle-like structures and ECM expression. Alginate gel culture is a promising new tool for the study of follicular morphogenesis, polarity, the expression pattern of ECM components and of the interaction between thyrocytes and ECM. It avoids interference caused by gels composed of ECM components.

Meprins, membrane-bound and secreted astacin metalloproteinases

The astacins are a subfamily of the metzincin superfamily of metalloproteinases. The first to be characterized was the crayfish enzyme astacin. To date more than 200 members of this family have been identified in species ranging from bacteria to humans. Astacins are involved in developmental morphogenesis, matrix assembly, tissue differentiation and digestion. Family members include the procollagen C-proteinase (BMP1, bone morphogenetic protein 1), tolloid and mammalian tolloid-like, HMP (Hydra vulgaris metalloproteinase), sea urchin BP10 (blastula protein) and SPAN (Strongylocentrotus purpuratus astacin), the 'hatching' subfamily comprising alveolin, ovastacin, LCE, HCE ('low' and 'high' choriolytic enzymes), nephrosin (from carp head kidney), UVS.2 from frog, and the meprins. In the human and mouse genomes, there are six astacin family genes (two meprins, three BMP1/tolloid-like, one ovastacin), but in Caenorhabditis elegans there are 40. Meprins are the only astacin proteinases that function on the membrane and extracellularly by virtue of the fact that they can be membrane-bound or secreted. They are unique in their domain structure and covalent subunit dimerization, oligomerization propensities, and expression patterns. They are normally highly regulated at the transcriptional and post-translational levels, localize to specific membranes or extracellular spaces, and can hydrolyse biologically active peptides, cytokines, extracellular matrix (ECM) proteins and cell-surface proteins. The in vivo substrates of meprins are unknown, but the abundant expression of these proteinases in the epithelial cells of the intestine, kidney and skin provide clues to their functions.

Local degradation of structural, mechanical, electrical and chemical properties of membrane electrode assembly in polymer electrolyte fuel cell

Polymer electrolyte fuel cell (PEMFC) is promising source of clean power in many applications ranging from portable electronics to automotive and land-based power generation. However, widespread commercialization of PEMFC is primarily challenged by degradation. The mechanisms of fuel cell degradation are not well understood. Even though the numbers of installed units around the world continue to increase and dominate the pre-markets, the present lifetime requirements for fuel cells cannot be guarantee, creating the need for a more comprehensive knowledge of material’s ageing mechanism. The objective of this project is to conduct experiments on membrane electrode assembly (MEA) components of PEMFC to study structural, mechanical, electrical and chemical changes during ageing and understanding failure/degradation mechanism. The first part of this project was devoted to surface roughness analysis on catalyst layer (CL) and gas diffusion layer (GDL) using surface mapping microscopy. This study was motivated by the need to have a quantitative understanding of the GDL and CL surface morphology at the submicron level to predict interfacial contact resistance. Nanoindentation studies using atomic force microscope (AFM) were introduced to investigate the effect of degradation on mechanical properties of CL. The elastic modulus was decreased by 45 % in end of life (EOL) CL as compare to beginning of life (BOL) CL. In another set of experiment, conductive AFM (cAFM) was used to probe the local electric current in CL. The conductivity drops by 62 % in EOL CL. The future task will include characterization of MEA degradation using Raman and Fourier transform infrared (FTIR) spectroscopy. Raman spectroscopy will help to detect degree of structural disorder in CL during degradation. FTIR will help to study the effect of CO in CL. XRD will be used to determine Pt particle size and its crystallinity. In-situ conductive AFM studies using electrochemical cell on CL to correlate its structure with oxygen reduction reaction (ORR) reactivity

Interface membrane fibroblasts around aseptically loosened endoprostheses express MMP-13

The objective of this article was to assess whether matrix metalloproteinase-13 (MMP-13) is produced by cells of the peri-implant interface tissues and to further characterize these cells. Tissue specimens were collected from the bone-prosthesis interface at the time of revision surgery of clinically loosened hip and knee arthroplasties (n = 27). Synovial tissues from osteoarthritic patients and young patients with mild joint deformity were used as controls (n = 6). Tissue samples were fixed in 4% PFA, decalcified with EDTA, and embedded in paraffin. Sections (4 microm) were stained with hematoxylin/eosin and for the osteoclastic marker enzyme tartrate resistant acid phosphatase. Monocytes/macrophages were characterized with a monoclonal antibody against CD68 and mRNAs encoding MMP-13 and alpha(1) collagen I (COL1A1) were detected by in situ hybridization. Cells expressing transcripts encoding MMP-13 were found in 70% of the interface tissues. These cells colocalized with a cell population expressing COL1A1 mRNA, and were fibroblastic in appearance. MMP-13 expressing cells were found in the close vicinity of osteoclasts and multinuclear giant cells. No signals for transcripts encoding MMP-13 were detected in multinuclear giant cells or in osteoclasts. Control tissues were negative for transcripts encoding MMP-13 mRNA. Fibroblasts of the interface from aseptically loosened endoprostheses selectively express MMP-13. By the expression and the release of MMP-13, these fibroblastic cells may contribute to the local degradation of the extracellular matrix and to bone resorption.

Monitoring protein-protein interactions between the mammalian integral membrane transporters and PDZ-interacting partners using a modified split-ubiquitin membrane yeast two-hybrid system

PDZ-binding motifs are found in the C-terminal tails of numerous integral membrane proteins where they mediate specific protein-protein interactions by binding to PDZ-containing proteins. Conventional yeast two-hybrid screens have been used to probe protein-protein interactions of these soluble C termini. However, to date no in vivo technology has been available to study interactions between the full-length integral membrane proteins and their cognate PDZ-interacting partners. We previously developed a split-ubiquitin membrane yeast two-hybrid (MYTH) system to test interactions between such integral membrane proteins by using a transcriptional output based on cleavage of a transcription factor from the C terminus of membrane-inserted baits. Here we modified MYTH to permit detection of C-terminal PDZ domain interactions by redirecting the transcription factor moiety from the C to the N terminus of a given integral membrane protein thus liberating their native C termini. We successfully applied this "MYTH 2.0" system to five different mammalian full-length renal transporters and identified novel PDZ domain-containing partners of the phosphate (NaPi-IIa) and sulfate (NaS1) transporters that would have otherwise not been detectable. Furthermore this assay was applied to locate the PDZ-binding domain on the NaS1 protein. We showed that the PDZ-binding domain for PDZK1 on NaS1 is upstream of its C terminus, whereas the two interacting proteins, NHERF-1 and NHERF-2, bind at a location closer to the N terminus of NaS1. Moreover NHERF-1 and NHERF-2 increased functional sulfate uptake in Xenopus oocytes when co-expressed with NaS1. Finally we used MYTH 2.0 to demonstrate that the NaPi-IIa transporter homodimerizes via protein-protein interactions within the lipid bilayer. In summary, our study establishes the MYTH 2.0 system as a novel tool for interactive proteomics studies of membrane protein complexes.

[Lower eyelid reconstruction of traumatically caused cicatricial entropion with amniotic membrane or mucosal membrane grafts]

BACKGROUND: A retrospective evaluation was undertaken of eyelid reconstruction with amniotic membrane or oral mucosal membrane transplantation in patients with lower lid cicatricial entropion after orbital surgery. PATIENTS AND METHODS: Seven patients (four women) were treated with a scar tissue dissection and an amniotic membrane or mucosal membrane transplantation between 2003 and 2006 (Five amniotic membrane grafts and two oral mucosal membrane grafts). In selected cases additional procedures like a lateral tarsal strip operation, a tarsal fracture, or the reinsertion of the lower lid retractors were performed. RESULTS: All patients showed a favourable postoperative result with a good anatomic correction of the entropion and a regression of the preoperative disturbances. All the grafts took well. Two patients had to be reoperated twice and one patient three times as a result of a relapse of the cicatricial entropion. However, as well in these patients the anatomical and functional result was favourable at the end. CONCLUSIONS: The difficult scar dissection with the subsequent amniotic membrane or oral mucosal membrane transplantation seems to be an appropriate procedure to reconstruct complicated cicatricial entropion after orbital surgery.

Characterization Of Porous Media In Proton Exchange Membrane Fuel Cell Based on Percolation Studies

Water management in the porous media of proton exchange membrane (PEM) fuel cells, catalyst layer and porous transport layers (PTL) is confronted by two issues, flooding and dry out, both of which result in improper functioning of the fuel cell and lead to poor performance and degradation. The data that has been reported about water percolation and wettability within a fuel cell catalyst layer is limited to porosimetry. A new method and apparatus for measuring the percolation pressure in the catalyst layer has been developed. The experimental setup is similar to a Hele-Shaw experiment where samples are compressed and a fluid is injected into the sample. Pressure-Wetted Volume plots as well as Permeability plots for the catalyst layers were generated from the percolation testing. PTL samples were also characterizes using a Hele-Shaw method. Characterization for the PTLs was completed for the three states: new, conditioned and aged. This is represented in a Ce-t* plots, which show a large offset between new and aged samples.

Tmem27: a cleaved and shed plasma membrane protein that stimulates pancreatic beta cell proliferation

The signals and molecular mechanisms that regulate the replication of terminally differentiated beta cells are unknown. Here, we report the identification and characterization of transmembrane protein 27 (Tmem27, collectrin) in pancreatic beta cells. Expression of Tmem27 is reduced in Tcf1(-/-) mice and is increased in islets of mouse models with hypertrophy of the endocrine pancreas. Tmem27 forms dimers and its extracellular domain is glycosylated, cleaved and shed from the plasma membrane of beta cells. This cleavage process is beta cell specific and does not occur in other cell types. Overexpression of full-length Tmem27, but not the truncated or soluble protein, leads to increased thymidine incorporation, whereas silencing of Tmem27 using RNAi results in a reduction of cell replication. Furthermore, transgenic mice with increased expression of Tmem27 in pancreatic beta cells exhibit increased beta cell mass. Our results identify a pancreatic beta cell transmembrane protein that regulates cell growth of pancreatic islets.